In recent years, home care and community health care in a doctor's office and a clinic have improved and the number of early diagnoses and the number of urgent laboratory tests have increased. Against this backdrop, analyzing devices have been demanded that can quickly and easily perform measurement with high accuracy even if users are not medical technologists. Thus small analyzing devices for POCT (Point of Care Testing) have received attention that can perform reliable measurement in a short time without complicated operations.
POCT is a generic name of inspections conducted in locations “close to patients”, for example, in consulting rooms of practitioners and specialists, hospitals, and clinics for outpatients. POCT has been a notable method that is useful for improving the quality of diagnoses such that a doctor quickly judges an inspection result, immediately performs treatment, and monitors the process of the treatment and the prognosis. Inspections conducted by such small analyzing devices can reduce the cost of transporting specimens, the cost of equipment, and the cost of unnecessary inspections, thereby reducing the total inspection cost as compared with inspections conducted in central examination rooms. In the U.S. featuring rational hospital management, a POCT market has rapidly expanded and is expected to grow worldwide, including Japan.
In a dry-type biosensor (analysis element) typified by an immuno-chromatographic sensor, an adjustment of a reagent is not necessary and a target analyte contained in a liquid sample can be analyzed only by a simple operation, e.g., dropping of the liquid sample (specimen solution) such as blood and urine to be measured into the biosensor. Currently, a large number of dry-type biosensors have been put into practical use as representative POCT biosensors because dry-type biosensors are quite useful for easily and quickly analyzing a target analyte in a liquid sample.
In an immuno-chromatographic sensor using an antigen-antibody reaction, detection involves high specificity and a strong binding force. Thus in an analysis of quite a low concentration of a bioactive substance, an immuno-chromatographic sensor demonstrates more excellent properties than other kinds of sensors. Such an immuno-chromatographic sensor includes, on a porous carrier such as nitrocellulose and glass fiber filter paper, a reaction portion in which a reagent to be bound specifically to a target analyte is immobilized; a labeled reagent retaining portion labeled with a reagent bound specifically to the target analyte and an immobilized reagent; and a sample adding portion in which a liquid sample is added to the labeled reagent retaining portion or upstream of the labeled reagent retaining portion in a sample extending direction. The following will discuss the measurement principle. First, the liquid sample is added to the sample adding portion (specimen solution supply portion). The added liquid sample is extended on the porous carrier and reaches the labeled reagent retaining portion. After that, the liquid sample is extended downstream, passes through a reaction layer, and is extended downstream on the porous carrier. In the case where the liquid sample contains a target analyte, the target analyte first reacts specifically with a labeled reagent and then is extended to the reaction portion. When reaching the reaction portion, the target analyte having reacted specifically with the labeled reagent also reacts specifically with the immobilized reagent of the reaction portion, resulting in a color reaction depending on the concentration of the target analyte. Thus by analyzing the color reaction, the concentration of the target analyte can be confirmed. By using this principle, many diagnostic agents of pregnancy, cancer markers, cardiac markers, and the like have been put into practical use and are available on the market. The measurement principle was described according to, e.g., a sandwich reaction. A competitive reaction may be used for measurement.
In many immuno-chromatographic sensors, quantitative decisions are mainly made. In recent years, some products have been developed to quantitatively measure the concentration of a target analyte according to the degree of a color reaction in a reaction portion by using a reader, like diagnostic agents of cardiac markers. An optical phenomenon is mainly used as a principle of quantitative measurement. In frequently used methods, a color is generated by interaction between a target analyte and a specific protein immobilized on a chromatographic carrier of a porous film and the intensity of the color is optically detected by a detector.
In the case of quantitative measurement using an immuno-chromatographic sensor, however, information is obtained only by optically reading the degree of a color in a reaction portion. Thus just a few pieces of information are obtained and it is difficult to conduct quantitative measurement with higher accuracy.
For this reason, various methods have been used in which the number of pieces of information is increased by electrochemical techniques. For example, Patent Literature 1 describes a biosensor that is an immuno-chromatographic sensor of lateral flow type. The biosensor is made up of an electrode part and a chromatography matrix part having an electrode formed of silver/silver chloride paste ink on a printing substrate that is pure cellulose chromatography paper.
In this method, the biosensor is fabricated by forming the electrode part on a preformed porous film by screen printing. In screen printing, a printing paste prepared with an organic solvent is frequently used. However, many porous films are deformed by the penetration of organic solvents, so that the porous shape may be broken. Therefore, the porous shape of the porous film may be broken by forming the electrode by screen printing and the porous film cannot act as expected. Thus the selection of a solvent is quite important in the printing of an electrode on a porous film.
Even when a selected paste material does not hamper printing, a printing paste coming inside a porous film may cause clogging, so that a specimen solution may be insufficiently extended or the specimen solution may not penetrate into a hydrophobic paste material. It is quite difficult to perform printing while avoiding these problems.
Further, in the case where an electrode is formed by screen printing, a paste bleeding in printing varies a measurement electrode area, disadvantageously causing variations in response characteristics. The occurrence of this problem is extremely high in Patent Literature 1 in which the electrode is formed on the porous film. Thus this serious problem should be solved to improve quantitative accuracy in the biosensors using carriers of porous films. Only measuring electrodes of equal areas may be selected but the selection of such measuring electrodes may reduce the yield, resulting in high cost.
In order to solve these problems, it is essential as an inspection process to measure an electrode area after printing on a porous film. The addition of the inspection process slightly improves the measurement accuracy of the biosensor but increases the number of steps and the manufacturing cost of the biosensor, so that even if the performance of sensors improves, it is impossible to provide inexpensive biosensors. For this reason, it has been strongly requested to provide inexpensive materials enabling electrochemical analyses with uniform performance. Another problem is that the formation of an electrode on a biosensor by screen printing makes it impossible to read an optical signal in a reaction portion, though an electrochemical measurement method can be realized.
Patent Literature 2 describes an electrode immune-response analyzing device of a flow-through type in which an upper cover having a sample inlet, an upper filter, first and second impregnating portions, a passage member, an electrode portion formed on an electrode substrate, a specific binding substance insolubilizing film, an absorbing portion, and a lower substrate are stacked in the thickness direction and a sample is passed in the thickness direction (stacking direction). In this method, the specific binding-substance insolubilizing porous film is not bonded but is stacked on an electrode formed by screen printing on the electrode substrate made of PET. The amount of a substance having undergone a specific binding reaction on the porous film is quantitatively measured by determining, on the electrode, the amount of current obtained from an enzyme-labeled antibody. Unlike in Patent Literature 1 in which the electrode is directly formed on the porous film, the electrode and the porous film are separately fabricated in this configuration and thus the porous film is not directly coated with a printing paste. It is therefore possible to prevent the porous film from being seriously broken, increase the selection of printing pastes for printing the electrode portion, and reduce the occurrence of variations in an electrode area in the event of blurred printing. However, unless the specific binding-substance insolubilizing porous film and the electrode that have been separately prepared are stacked with high accuracy, it is not possible to achieve high-accuracy measurement. Further, in this system, measurement is conducted by reading an electrochemical signal obtained by an enzyme reaction and thus a two-step reaction is necessary in which the amount of a substance having undergone a specific binding reaction is read by the enzyme reaction. Consequently, the quantitative accuracy may be affected by two factors that are variations caused by a specific binding reaction and response variations caused by an enzyme reaction.
Patent Literature 3 describes a biosensor including a porous thin film on the surface of an electrode. In this biosensor, a collected whole blood sample is chromatographically moved by the porous thin film to separate plasma from the whole blood and only the plasma is measured in contact with an electrode system. Since only the plasma is separated, the influence of hematocrit in blood is eliminated. In measurement, however, a sufficient time is necessary between the separation of the plasma and the contact with the electrode system and the accuracy of separation is not enough to fully separate the plasma. Thus the quantitative accuracy is not so improved, though the influence of hematocrit is reduced.
Citation List
Patent Literature
    Patent Literature 1: International Publication No. WO01/004614    Patent Literature 2: Japanese Patent Laid-Open No. 8-75748    Patent Literature 3: Japanese Patent Laid-Open No. 2002-90331